WO1996006942A1

WO1996006942A1 - Genetically altered t-cells

Info

Publication number: WO1996006942A1
Application number: PCT/EP1995/003410
Authority: WO
Inventors: Hartmut Wekerle; Hans Thoenen; Rainer Kramer; Yiping Zhang
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1994-08-31
Filing date: 1995-08-30
Publication date: 1996-03-07
Also published as: AU3474595A; CA2198891A1; EP0782627A1; JPH10505235A

Abstract

The invention concerns genetically altered mature T-cells with autoreactive receptors which carry in their genome at least one externally introduced gene which can express a (poly)peptide. The invention further concerns drugs containing such T-cells.

Description

Genetically modified T cells

The invention relates to genetically modified mature T cells with autoreactive receptors which carry at least one externally introduced gene in their genome which is capable of expressing a (poly) peptide. The invention further relates to medicaments which contain such T cells.

Modern medicine continues to develop in order to avoid heavy surgical interventions and to achieve clinically equivalent or more favorable results by means of other, new procedures which, above all, free the patient from the physical trauma of an operation.

Such new methods have been developed in the past, for example on the basis of knowledge of molecular biology, in particular in connection with the disciplines of immunology and neurology. For example, attempts have been made to treat tumor therapies by applying tigen directed antibodies, coupled to toxins to establish; see. Vallera, Blood 83: 309-317 (1994). On the other hand, when isolating and cloning neurotrophic factors, such as BDNF, NGF or NT-3, it has been considered to use them for the treatment of degenerative diseases of the nervous system (cf., for example, WO 91/03568).

Many of these methods are based on the idea of transporting an active ingredient to the desired destination in such a way that it only develops its activity there. The practical implementation of this idea has so far generally failed because the active ingredients do not actually reach their destination, or only to a certain percentage. Some tissues, especially the brain and the peripheral nerves, are separated from the bloodstream by a dense endothelial blood-brain barrier. Macromolecular agents that are administered into the blood are therefore very difficult to reach the "sequestered" tissues. On the one hand, the desired therapeutic effect of the methods mentioned may not be achieved, on the other hand, as in the case of immunotoxins, undesirable side effects may occur if the active substance is unloaded at a destination other than the desired one . As is known in the prior art, access for active substances known in the prior art, such as e.g. Immunotoxins, prevented by the blood-brain barrier. Exceptions for medically effective substances that could overcome this barrier have so far only been lipophilic substances or substances transported via specific receptors (e.g. transferrin).

The object of the invention was therefore to provide a vehicle with which a biologically active molecule can be transported to a desired target molecule or target tissue and only interacts with it. This problem is solved by providing a T cell with the features of claim 1. The invention thus relates to a genetically modified, mature T cell with the following properties:

(a) their receptor is autoreactive; and

(b) it carries in its genome at least one externally introduced gene which is normally not expressed in mature T cells and which is capable of expressing a (poly) peptide.

The term "genetically modified" in the sense of this application means that the T cell or its precursor has been modified using molecular biological techniques. Suitable molecular biological techniques, such as vector constructions, transformations or transfections or (inducible) expressions are described, for example, in Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring Harbor (1989).

The term "autoreactive" means that the T cell receptor bind an autoantigen presented by an MHC molecule to a suitable cell.

For the purposes of this invention, the term “externally introduced gene which is normally not expressed in mature T cells” means that the gene coding for the (poly) peptide, which may be of homologous or heterologous origin, does not exist in vivo in mature T cells is expressed and the expression is only achieved through the molecular biological manipulations. The externally introduced gene can, for example, be under the control of its own regulatory elements. According to the invention, one or more of these genes on one or more vectors may have been introduced into the T cells simultaneously or in succession.

With the T cell according to the invention, a vehicle is provided for the first time which can transport and deliver a heterologous protein directly and specifically to a target sequence or target location, for example in the nervous system. This specificity is achieved by the specificity of the T-cell receptor, which recognizes a certain autoantigen presented by an MHC molecule. Since it is known that If the reactivity pattern of a T cell receptor is highly specific, the T cell receptor and thus the molecule to be transported do not interact with other undesired molecules in the body of a patient. In the area of the nervous system there is another peculiarity that can be used for the proposed therapeutic route. While the cells of the healthy brain tissue hardly produce any MHC molecules and are therefore not able to present the (self) antigen recognizable to the T lymphocytes, the MHC antigens are induced in a large number of pathological changes. This relates in particular to degenerative and inflammatory processes, in the course of which glial cells of the nerve tissue acquire the ability to present antigens. In addition to the (auto) immune infections, diseases such as Parkinson’s disease or Alzheimer are examples. As has been shown, the brain-reactive T lymphocytes according to the invention preferably detect such altered brain areas, tissue areas where the therapeutic agents are particularly needed.

The ability of activated T cells to penetrate the blood-brain barrier was thus exploited in the present invention.

In medical practice, a pool of mature T cells is removed from a patient for producing the T cell according to the invention by customary methods and immediately selected for uniform properties, in particular with regard to the receptor specificity. After the molecular biological manipulations have been carried out, the T cell according to the invention is usually reapplied to the patient from whom the T cells were originally removed in order to avoid histocompatibility problems.

With the system according to the invention, neurotrophic molecules or anti-inflammatory factors can, for example, be brought to specific target locations and used there to prevent harmful side effects. This can take place, for example, in that the neurotrophic factors act on a target cell and there a cascade is desired. biochemical or metabolic events. An example of this is that in Parkinson's patients the remaining substantia nigra is kept alive and thus further cell loss is avoided.

In a preferred embodiment of the T cell according to the invention, the expressed (poly) peptide is exposed or secreted on the surface.

The expression of the (poly) peptide on the surface occurs when the (poly) peptide has a membrane anchoring sequence. If the (poly) peptide does not naturally contain such a sequence, it can be added genetically using conventional methods without any problems. The surface expression of the (poly) peptide is particularly desirable if an interaction is only to take place with the cell to which the T cell has docked via its receptor.

In contrast, the secretion of the (poly) peptide is desirable if its effect is not to be restricted to the docked cell itself. Examples of secreted (poly) peptides are antibodies or neurotrophic, immunomodulating or growth-regulating substances. If these (poly) peptides naturally do not have any signal sequences necessary for export, the person skilled in the art can easily attach them by genetic engineering.

In a further preferred embodiment of the T cell according to the invention, the expressed (poly) peptide inhibits or activates the expression of endogenous proteins, the inhibition or activation leading to a change in the pattern on the surface of endogenous (poly) peptides or secreted proteins / Peptides.

In this embodiment of the invention, the (poly) peptide has a trigger function in order to change the expression pattern of the cell. This change in the expression pattern in turn exerts a desired effect on a target molecule or a target cell, for example via the Expression or the inhibition of the expression of a neurotrophic, immunomodulating or growth-regulating molecule or a surface molecule is achieved. An example of this embodiment are immunomodulators which regulate the expression of the MHC molecules down in the brain.

In a further preferred embodiment of the T cell according to the invention, its receptor is directed against an epitope of the nervous system.

In a particularly preferred embodiment of the invention, the epitope is an epitope of the central nervous system. Examples include proteins that occur specifically in certain brain areas or defined cell types. This is the case for transmitter-specific enzymes (tyrosine hydroxylase), but also possibly for the pigment of the substantia nigra (Parkinson) or certain transmitter receptors. Regionally specific receptors and neuropeptides, e.g. Galanin to call CGRP.

In a further particularly preferred embodiment of the invention, the epitope is an epitope of the peripheral nervous system.

Another particularly preferred embodiment of the invention relates to a T cell, the receptor of which is directed against an epitope of the myelin protein P2 or against S100.

In a further preferred embodiment of the T cell according to the invention, the epitope is an epitope of a state-specific protein, for example amyloid precursor protein (APP), a tissue-specific protein, a receptor protein, for example regionally specific GABA, glutanate or glycine Receptors, a cytoskeleton protein or a transmitter synthesis enzyme, for example tyrosine hydroxylase. A further preferred embodiment of the invention relates to a T cell, the expressed (poly) peptide being a neurotrophic factor.

The term "neurotrophic factor" is understood to mean mediators who control the survival, growth and differentiation of neuronal cells. Neurotrophins also have the ability to protect neurons against various types of damage and to restore impaired functions.

In a particularly preferred embodiment of the T cell according to the invention, the neurotrophic factor belongs to the NGF gene family (neurotrophins) and is, for example, NGF, BDNF, NT-3, NT-4/5, NT-6 or other neurotrophic factors, such as FGF , GDNF or CNTF. The transport of these neurotrophic factors, alone or in combination, by means of inventive autoreactive T cells directed against an epitope of the nervous system enables e.g. the study and application of therapeutic options for degenerative, traumatic, metabolic or myelodegenerative diseases of the nervous system, such as Alzheimer's disease, Parkinson's see disease, tumors, degenerative diseases, autoimmune diseases, infections, metabolic or traumatic diseases.

A further preferred embodiment of the invention relates to a T cell which has the CD4 ⁺ CD8 ^~ phenotype. T-helper cells will primarily be used as vehicles when CD4 ⁺ T lymphocytes occur in two subclasses (Thl and Th2), which differ in the spectrum of the cytokines produced and thus also differ Have functions. In addition to IL-2, Thl cells produce especially interferon-gamma and tumor necrosis factor (-α and -ß). Th2 cells, on the other hand, specialize in IL-4, IL-5 and IL-10. Thl cells would mainly be used in degenerative processes, but also in tumors (they induce MHC antigens), while Th2 cells are inflammatory have a dampening effect and would be helpful for autoimmune diseases (e.g. MS etc.).

Another preferred embodiment of the invention relates to a T cell which has the CD4 ^" CD8 ⁺ phenotype. CD8 ⁺ lymphocytes are mostly highly effective killer lymphocytes which are used in the field of tumor and virus defense can.

In a further preferred embodiment of the T cell according to the invention, the externally introduced gene is under the control of retroviral regulatory elements. “Retroviral regulatory elements” is understood here to mean any regulatory element for the expression of genes which has been isolated from retroviruses or which has been derived from such elements. Examples of such regulatory elements are retroviral promoters or enhancers. The use of retroviral regulatory elements has the particular advantage that the genes under their control are only expressed when the T cells are activated. In other words, the (poly) peptide is only expressed when the T cells are activated by interaction of the receptor with the antigen. In this way, tissue-specific expression and controllability of the expression of the (poly) peptide is achieved by activating the T cell at the target site. As is known to the person skilled in the art, the regulatory elements can be adapted to the respective requirements.

A further preferred embodiment of the invention relates to a T cell in which the externally introduced gene is under the control of an inducible promoter. The use of an inducible promoter also has the advantage that the expression of the (poly) peptide in the T cell can be controlled and can therefore be tissue-specific. Inducible promoters are widely known in the art. In a further preferred embodiment of the T cell according to the invention, the externally introduced gene is under the control of T cell-specific regulatory elements. Here, a more finely tuned activation of the externally introduced gene (transgene) could be achieved, possibly also a longer (permanent) expression of the transgene.

In a further preferred embodiment of the T cell according to the invention, the externally introduced gene is recombinant components of the genome of a lytic / lysogenic T cell-specific virus, which can lyse the T cell after the receptor has been attached to the target epitope . This embodiment also serves for the defined release of the (poly) peptide at its destination. The function of the secretion from the cell in the embodiments of the invention discussed above is enhanced by the apoptosis of the T-lymphocytes according to the invention, which are promoted by the special microenvironment of the target tissue.

The invention further relates to a medicament which contains a T cell according to the invention in combination with a pharmaceutically acceptable carrier.

The formulation of such drugs is known to the person skilled in the art.

In principle, the medicaments according to the invention can be used for all diseases whose treatment has a local interaction of the (poly) peptide or a combination of (poly) peptides with one or more target molecules. It is conceivable that such a drug contains one or more different T cells according to the invention, which in turn express one or more of the externally introduced (poly) peptides. The pharmaceuticals according to the invention are suitable, for example, for the treatment of regenerative or inflammatory diseases. Finally, the invention relates to the use of an inventive T-cell for the treatment of degenerative Erkran¬ effects such as ALS (amyotrophic lateral sclerosis), Alzheimer's disease, pressures Parkinson ¹ shear's disease, multiple sclerosis, trauma, infections, autoimmune diseases, immunosuppression, vascular insults, tumors, or acute edema.

The figures show:

FIG. 1A: retroviral NGF construct which has been used for transduction of R4 lymphocytes; B: NGF production by R4 T cells after retroviral

Transduction; C: NGF expression in EAN (experimental autoimmune neuritis) in filtrates by transduced T-lymphocytes (in situ hybridization). The animals were sacrificed 14 days after the injection of 2.5 × 10 ⁶ NGF-transduced (upper and lower diagram) or wild-type R4 lymphocytes (middle diagram) into the tail vein of ether-anesthetized adult Lewis rats ;

Fig. 2 Clinical results and weight changes in rats in which EAN has been induced. The animals were examined daily for clinical disease symptoms (FIG. 2A) and change in weight (FIG. 2A);

3 Histopathological changes in the sciatic nerve after injection of wild type (a), sham-transduced (b) or NGF-transduced R4 lymphocytes (c).

Left row: toluidine blue staining of semi-thin sections. Right row: electron micrograph.

Fig. 4 Cellular composition of EAN infiltrates caused by NGF-transduced or control T cell lines in the presence or absence of exogenous NGF: Immunohistochemical staining of W3 / 13-positive T lymphocytes (left row) or EDI-positive macrophages (right row) in the sciatic nerve, positive cells. Positive cells are cells which bind the antibody, ie which express the marker antigen on the surface.

Treatment groups: a, only wild-type R4 cells __. Ι, b, NGF-transduced R4 cells, c) and d) Wilc, -p-R4 cells in combination with the administration of either BSA (c) or NGF protein (d), e, distribution of ET1- and W3-13 positive cells a..ιs a) to d) shown as a bar graph, including the values for recipients of sham-translated R4 cells (immunocytochemistry not shown). * Statistical significance was examined by the Student t test p <0.00001.

The examples illustrate the invention.

example 1

Transduction of P2-specific, MHC class II-restricted CD4 ⁺ T lymphocytes

The neuritogenic, P2 protein-specific R4 cell line was used as a cellular vehicle of NGF in the peripheral nervous system. R4 lymphocytes induce demyelizing autoimmune inflammation (EAN), which is limited only to the peripheral nervous system (PNS) (Linington et al., J. Immunol. 133 (1984), 1946-1950). The lesion is characterized by a strong infiltration of mononuclear cells, followed by extensive destruction of the myelin sheath (Izumo, S. et al., Lab. Invest. 53 (1985), 209-218). The time course and the extent of the pathogenic reactions are highly predictable. The clinical manifestations of this disease develop very quickly four days after the induction of the T cells and are caused by one markedly reduced nerve conduction within a few hours (Heiniger, K. et al., Ann. Neurol. 19 (1986), 44-49).

For the transport of NGF to the target molecule, R4 lymphocytes were transduced with a replication-deficient, ecotrophic recombinant retrovirus, which encodes the cDNA of mouse NGF (FIG. 1 a). To construct this vector, an 882 bp Sphl / PstI fragment of the mouse NGF cDNA (J. Scott et al., Nature 302 (1983), 538-540) was first under the control of the viral promoter in the 5'-LTR of the 5.8 kb vector pLXSN (D. Miller et al., Bio Technique 7, (1989) 980-990). Functional splice donor (SD) and splice acceptor (SA sites) are located upstream of the NGF cDNA. An internal SV40 promoter controls the expression of the G418 resistance gene (neo), while the polyadenylation (pA) takes place within the 3 'LTR. The initiation of the transcription is shown by arrows. With the construct thus obtained, the GP + E-86 packaging cell line (D. Markowitz et al., J. Virol. 62 (1988) pp. 1120-1124) was transfected by calcium phosphate coprecipitation. The retrovirus-containing supernatant was collected after 48 hours and used to infect a GP + E-86 culture treated with tunicamycin (100 ng / ml 16 hours). Individual G418 (1 mg / ml) resistant clones were subcloned after 12 days and tested for the production of infectious retroviral particles, the number of G418-resistant NIH 3T3 fibroblasts being determined. The titers of the individual packaging clones were in the range from 2 to 6 x 10 plaque-forming units (pfu) per ml.

R4 lymphocytes were activated for 48 hours in the presence of native bovine myelin P2 protein (20 μg / ml) and thymus cells (1x10 cells / ml irradiated at 2000 R) as a source of antigen-presenting cells. These stimulated lymphoblasts were treated with RPMI 1640 containing 5% fetal calf serum, 15% T cell growth factor (Supernatant from ConA-activated mouse spleen cells) supplemented with 80% retrovirus ( ⁶ × 10 ⁶ pfu / ml) and 8 μg / ml polypene transduced. Virus-containing supernatant was removed by repeated washing and transduced R4 lymphocytes were resuspended in RPMI 1640 medium and grown in an atmosphere containing 5% CO2 at 37 ° C. Conditioned media of 10 wild-type (R4) -, NGF-retrovirus transduced (R4NGF) - or sham-transfected (retrovirus, which does not encode NGF; R4mock) R4-lymphocytes were found at NGF concentration (pg / ml) 12 hours after the Transfection tested by an immunoassay testing two binding sites (S. Korsching et al., Proc Natl. Acad. Sci. USA 80 (1983) 3513-3516). The result of this experiment shows that wild-type R4 cells and R4 cells which were mock-transfected did not produce any detectable amounts of NGF. In contrast, R4 lymphocytes transduced with the NGF retrovirus separated more than 2000 pg / ml NGF the culture medium (Fig. lb). NGF production by R4 cells is comparable to that of genetically modified rat fibroblasts, which were used by transmission into the central nervous system to provide NGF for the treatment of experimentally produced neurodegenerative diseases (MD Kawaja et al., J. Neurosci. 12: 2849-2864 (1992). The transduction itself did not change the antigen specificity or the proliferative activity of the R4 cells; the Thl-like secretion pattern on cytokines with a strong production of 11-2 and interferon-7 also remained unchanged (results not shown).

Example 2

In vivo migration of neurotogenic T cells transduced with the NGF gene

The transduced R4 lymphocytes infiltrated the peripheral nervous system to the same extent as wild-type or sham-transfected R4 cells (FIG. 4). 14 days after the travenous injection of NGF-transduced R4 cells could be shown by in situ hybridization (Fig. lc) that these genetically modified lymphocytes still strongly express NGF-mRNA in vivo in the peripheral nervous system. For the injection, 2,5 × 10 ⁶ -stimulated wild-type (R4), NGF-transduced (R4NGF) or sham-transfected (R4mock) -R4 lymphocytes into the tail vein were ether-anesthetized Lewis rats in a total volume of 1 ml injected on day 0. Clinical symptoms were recorded according to the following classification: 1: partial loss of tail tone; 2: complete loss of tail tone; 3: paraparesis of the hind legs; 4: Paralysis of the hind legs; 5: Paraparesis of the front legs. All values are given here as mean values of each group (n = 3), standard deviations between day 0 and day 9 being included.

The animals were sacrificed 14 days after the injection of 2.5x10 R3-T lymphocytes and perfused with 4% paraformaldehyde containing 0.5% glutardialdehyde in 0.1 M phosphate-buffered saline, using the same fixative overnight at 4 ° C immersion fixed and then embedded in Epon by conventional methods. 1 µm sections were made and stained with 0.1% toluidine blue to estimate the extent of demyelination; the unit of measure here is 2.5 μm. Ultra-thin sections were made and prepared for electron microscopy, as described by J. Gehrmann et al. Lab. Invest. 67 (1992) 100-113; one unit on the scale is 1 μm.

Bovine serum albumin or NGF was administered to ether-anesthetized Lewis rats by implantation of a sponge gel cushion (spongostan) containing either 125 μg NGF (2.5 S) or 250 μg BSA (Sigma, Cohn fraction V) in 50 μl 0.9% Na ¬ trium chloride contained. The administration took place in the vicinity of the left or right sciatic nerve in the sub-gluteal region of the same rat 24 hours before the injection of 2.5 × 10 ⁶ wild-type R4 cells. EDI and W13 positive Cells were counted from five areas of each preparation (n = 3) and the values were given as mean values including the standard deviation in cells per mm ² .

For the in situ hybridizations, cryostat sections (10 μm) of the sciatic nerve preparations (cut lengthwise) were carried out under stringent conditions either with single-stranded ^s 35-labeled antisense (upper and middle diagram) or more meaning-oriented (lower diagram) ) NGF-specific cDNA probes hybridized by reverse transcription from sense or antisense NGF cRNA according to customary methods (E. Castren et al., Proc. Natl. Acad. Sci. USA 89 ( 1992) 9444-9448).

Example 3

Anti-inflammatory effects of locally released NGF

The morphological changes caused by NGF-transfected R4 cells differed significantly from typical EAN, which was brought about by non- or sham-transfected R4 cells (FIG. 3). The demyelination of the sciatic nerve axons V / L r 14 days after the transfer of NGF-synthesizing R4 lymphocytes was drastically reduced (FIG. 3c), whereas that with the wild type (FIG. 3a) or with the sham-transfected R4 cells (FIG 3b) treated animals showed the typical changes in peripheral myelin sheaths. The immunocytochemical stains with the T-lymphocyte marker W3 / 13 showed no significant differences between the number of wild-type or of genetically modified R4 lymphocytes which infiltrated the sciatic nerve (FIG. 4e). In striking contrast to the classic EAN lesions, however, extremely few macrophages were detected after transfer with NGF-secreting R4 cells, the lysosomal marker EDI serving as the detection system (FIG. 4e). The changed pathogenicity of genetically modified NGF-producing T cells can theoretically have two reasons:

(a) The genetic modification per se could have reduced the pathogenic intrinsic potential of the neuritogenic T cells, or

(b) the NGF synthesized by the genetically modified T cells could have caused the strong reduction in demyelination.

If locally released NGF is responsible for the reduced pathogenicity, exogenously administered NGF should have a similar effect on macrophage activation. In fact, the local administration of NGF protein by implantation of a sponge gel cushion in vivo showed the same reduction in macrophage invasion as the NGF-producing R4 cells (FIG. 4d). The implantation of a sponge gel cushion containing BSA on the contralateral side (FIG. 4c), however, had no influence on the typical macrophage recruitment in EAN induced by the transfer of wild type R4 cells. The number of W3 / 13 positive cells on the differently treated sides of the rats were the same (FIGS. 4c, d).

Claims

Patent claims

1. Genetically modified, mature T cell with the following properties:

(a) their receptor is autoreactive; un ^"

2. T cell according to claim 1, wherein the expressed (poly) peptide is exposed or secreted on the surface.

3. T cell according to claim 1, wherein the expressed (poly) peptide inhibits or activates the expression of endogenous proteins, the inhibition or activation leading to a change in the pattern on the surface of expressed endogenous (poly) peptides or secreted (poly) peptides.

4. T cell according to one of claims 1 to 3, wherein the receptor is directed against an epitope of the nervous system.

5. T cell according to claim 4, wherein the epitope is an epitope of the central nervous system.

6. T cell according to claim 4, wherein the epitope is an epitope of the peripheral nervous system.

7. T cell according to claim 6, wherein the epitope is an epitope of the myelin protein P2 or S100.

8. T cell according to one of claims 1 to 4, wherein the epitope is an epitope of a state-specific protein, APP, a tissue-specific protein, a receptor proteins, a cytoskeleton protein or tyrosine hydroxylase.

9. T cell according to one of claims 1, 2 and 4 to 7, wherein the expressed (poly) peptide is a neutrophic factor.

10. T cell according to claim 8, wherein the neurotrophic factor is NGF, BDNF, NT-3, NT-4/5 and NT-6.

11. T cell according to one of claims 1 to 10, which has the CD4 ⁺ CD8 ^" phenotype.

12. T cell according to one of claims 1 to 10, which has the CD4 ^" CD8 ⁺ phenotype.

13. T cell according to one of claims 1 to 12, wherein the externally introduced gene is under the control of retroviral regulatory elements.

14. T cell according to one of claims 1 to 12, wherein the externally introduced gene is under the control of an inducible promoter.

15. T cell according to one of claims 1 to 12, wherein the externally introduced gene is under the control of T cell-specific regulatory elements.

16. T cell according to one of claims 1 to 12, wherein the externally introduced gene is a recombinant component of the genome of a lytic / lysogenic T cell-specific virus which the T cell after the receptor has attached to the target epitope can lyse.

17. Medicament containing a T cell according to one of claims 1 to 16 in combination with a pharmaceutically acceptable carrier.

18. Use of a T-cell suppressions according to one of claims 1 to 17 for the treatment of degenerative disorders, lesions, trauma, infections, autoimmune diseases, immune, tumors, ALS, vascular insults, Alzhei¬ mer's disease, Parkinson's disease ^1, multiple Sclerosis or acute edema.